My long term goals are (1) to define functional Xist sequences that govern the initiation and maintenance of X inactivation by systematic mutational analysis and (2) to define the transcriptional control mechanism wherein the presence of two Xist loci on homologous chromosomes results in only one allele expressing Xist RNA and inactivating the chromosome. I previously generated a line of Xist "knockout" mice and showed that the Xist gene (1) is required in cis for the initiation of X-inactivation, and (2) is essential for the selection mechanism that decides which X chromosome is inactivated. I have obtained evidence that my Xisdelta mutation on one allele alters the transcription state of the other (wild type) allele in Xist+/Xistdelta cells and propose to further characterize these changes. Based on other examples of gene regulation, I hypothesize that trans-interactions between the juxtaposed Xist alleles forces different transcription factor complexes to assemble at each Xist locus. Only one complex drives Xist RNA transcription causing heterochromatin to assemble throughout that X chromosome and be maintained inactive by Xist RNA and as well as other mechanisms. I propose to functionally localize the DNA and RNA sequences that are essential for the initiation of X-inactivation with an array of mutations introduced into XX female ES cells using the cre/Lox-based recombination mediated cassette exchange (RMCE) technique. The mutant ES cells are induced to undergo X-inactivation and assayed for X-inactivation defects using established techniques: immunolocalization for chromatin proteins, RNA FISH for Xist RNA coating, and the allele-specific SNuPE RT-PCR to measure the gene expression levels from the targeted X chromosome. I also propose to perform RMCE mutagenesis on the expressed Xist locus in fibroblasts where the endogenous homologous recombination machinery is inefficient. Analysis in fibroblasts allows one to define the sequences important for the maintenance of X-inactivation. Finally, I propose to test, using 3D FISH, whether the two Xist loci in a female cell homologously pair as I hypothesize.